Unraveling the intercellular communication disruption and key pathways in Alzheimer’s disease: An integrative study of single-nucleus transcriptomes and genetic association

Background Recently, single-nucleus RNA-seq (snRNA-seq) analyses have revealed important cellular and functional features of Alzheimer’s disease (AD), a prevalent neurodegenerative disease. However, our knowledge regarding intercellular communication mediated by dysregulated ligand-receptor (LR) interactions remains very limited in AD brains. Methods We systematically assessed the intercellular communication networks by using a discovery snRNA-seq dataset comprising 69,499 nuclei from 48 human postmortem prefrontal cortex (PFC) samples. We replicated the findings using an independent snRNA-seq dataset of 56,440 nuclei from 18 PFC samples. By integrating genetic signals from AD genome-wide association studies (GWAS) summary statistics and whole genome sequencing (WGS) data, we prioritized AD-associated Gene Ontology (GO) terms containing dysregulated LR interactions. We further explored drug repurposing for the prioritized LR pairs using the Therapeutic Targets Database. Results We identified 316 dysregulated LR interactions across six major cell types in AD PFC, of which 210 pairs were replicated. Among the replicated LR signals, we found globally downregulated communications in astrocytes-to-neurons signaling axis, characterized, for instance, by the downregulation of APOE-related and Calmodulin (CALM)-related LR interactions and their potential regulatory connections to target genes. Pathway analyses revealed 60 GO terms significantly linked to AD, highlighting Biological Processes such as ‘amyloid precursor protein processing’ and ‘ion transmembrane transport’, among others. We prioritized several drug repurposing candidates, such as cromoglicate, targeting the identified dysregulated LR pairs. Conclusions Our integrative analysis identified key dysregulated LR interactions in a cell type-specific manner and the associated GO terms in AD, offering novel insights into potential therapeutic targets involved in disrupted cell-cell communication in AD.


Background
Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects over 32 million individuals worldwide, resulting in substantial societal and economic burden [1].AD is characterized by extracellular deposits of β-amyloid, intraneuronal accumulation of neuro brillary tangles, ultimately resulting in neuronal death [2].Despite extensive research, with the exception of the amyloid deposition mechanism, the molecular and cellular mechanisms underlying AD remain elusive, which translates into limited effective therapies.
A delicate balance of the intercellular communications among non-neuronal and neuronal cells is essential for maintaining tissue homeostasis and normal brain functions such as synaptic pruning and synaptogenesis [3][4][5].Experimental and genetic evidence implicates the aberrant activation of microglia and astrocytes as contributing factors in the pathogenesis of neurodegenerative diseases.These activated cells exert downstream effects on neurons, further implicating them in diseases including Alzheimer's Disease (AD) [6,7].Recent studies have leveraged single-nucleus RNA sequencing (snRNAseq) data [8][9][10] and intercellular communication analysis tools [11,12] to identify complex intercellular communication within the postmortem AD brains [13][14][15].Most ndings suggest that microglia may contribute to AD's pathogenesis through ligand-receptor (LR) axis communication alterations [13][14][15], but the role of astrocytes in perturbed interactions remains largely unexplored.Hence, a more thorough intercellular communication analysis using snRNA-seq data is crucial for deeper insights into the interplay between non-neuronal and neuronal cells in AD.
Understanding the biological relevance of dysregulated intercellular signals in AD requires a comprehensive assessment of their associated pathways [4].An integrative analysis framework incorporating genetic variant data could enhance our understanding of biological pathways involving dysregulated communication signals in AD.Researchers have gained insight into atherosclerosisassociated biological mechanisms through snRNA-seq data-guided pathway-level polygenic scores (PGSs) analysis by integrating genome-wide association studies (GWASs) statistics and genotyping data [16].However, this integrative approach has not yet been applied to AD snRNA-seq data.While previous snRNA-seq studies have mapped AD GWAS risk loci [17][18][19][20] to AD-associated genes and open chromatin regions of microglia, astrocytes and oligodendrocytes [10,21,22], a more systematic integrative analysis is necessary to unveil the complex connections between genetic variants and speci c pathways encompassing dysregulated communication signals in AD.
To address this gap, we engineered a comprehensive integrative analysis framework to reconstruct the dysregulated intercellular communication network and identify their underlying biological functions in AD.
Speci cally, we collected two human prefrontal cortex (PFC) snRNA-seq datasets from AD individuals and healthy controls of two independent cohorts, used as discovery and replication datasets [8,10].Through a systematic comparative intercellular communication analysis, we identi ed dysregulated LR pairs and their potential target genes across six major cell types, namely astrocytes, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, and oligodendrocyte precursor cells (OPCs).Secondly, we conducted pathway-level analyses, leveraging GWAS statistics and genotyping data of AD participants and healthy controls, to prioritize biological pathways containing dysregulated communication signals.Lastly, our drug repurposing analysis, utilizing publicly available databases, revealed known and novel repurposable drugs for AD treatment.This study provides novel insights into the complex intercellular communication in AD postmortem brains, suggesting potential molecular mechanisms and therapeutic strategies for AD.

snRNA-seq data for AD
We collected two snRNA-seq datasets for AD research, one for discovery and the other for replication.The discovery dataset comprised postmortem human brain samples from 48 participants, sourced from The Religious Orders Study and Memory and Aging Project (ROSMAP) cohort.This dataset included 24 AD patients with mild to severe β-amyloid and other pathologies, and 24 sex/age of death-matched control subjects exhibiting no or minimal pathology [8].Droplet-based snRNA-seq data were generated from the PFC region of these 48 samples, resulting in transcriptome pro les for 80,660 single nuclei [8].The count matrix, mapped by using Cell Ranger (v.2.0.0,GRCh38.p5reference genome), was downloaded from the AD Knowledge Portal [23].
The independent replication dataset consists of postmortem human brain samples from 11 AD participants and 7 healthy controls, all from the University of California Irvine Institute for Memory Impairments and Neurological Disorders (UCI MIND) Alzheimer's Disease Research Center (ADRC) [10].The diagnosis of AD was de ned based on the Braak and plaque staging [10].The snRNA-seq data were generated from 61,472 isolated nuclei from the PFC region of these 18 individuals.We retrieved the data from the National Center for Biotechnology Information Gene Expression Omnibus (GSE174367).

snRNA-seq data quality control and annotation
We implemented universal preprocessing and quality control procedures for both the discovery and replication snRNA-seq datasets, starting from the count matrix, using the standard Seurat pipelines (v.4.3.0)[24].Speci cally, we retained cells containing between 200 and 6,000 features, with mitochondrial reads constituting less than 5% of the total reads.We then applied the standard lognormalization work ow to the gene expression matrix via the NormalizeData function in Seurat.Dimensionality reduction was executed using the Uniform Manifold Approximation and Projection (UMAP) technique, and visual representation was con ned to the initial two dimensions.We assigned cellular labels to eight major cell types in the brain, namely astrocytes, endothelial cells, excitatory neurons, inhibitory neurons, microglia, oligodendrocytes, OPCs, and pericytes, based on statistical enrichment of marker gene sets as delineated in the original publication [10,24].Endothelial cells and pericytes were excluded from the analysis due to their low cell count in both datasets.

Intercellular communication analysis
To infer the dysregulated intercellular communication signals between different cell types in the snRNAseq data, we used CellChat v.1.6.0 [11] based on an updated consensus LR dataset reported by Dimitrov et al. [12].The updated dataset contains 4,701 LR pairs compiled from 16 intercellular communication inference resources [12].
We followed the o cial pipeline provided by the developers to identify AD-speci c signaling.Accordingly, the intercellular communication probability of each LR pair between two cell populations within each condition (AD and control) was separately modeled and calculated.This calculation integrated the ensemble average gene expression per cell group and the consensus LR dataset, using the computeCommunProb function.Permutation tests were used to recognize statistically signi cant intercellular communications.To identify up-and down-regulated signaling LR pairs between AD and control, we used the netVisual_bubble function with the default parameters.An LR interaction between two cell types was considered to be context-speci c if it had permutation test p-value < 0.05 under the corresponding condition and it exhibited different communication probabilities compared to the alternate condition.Further ltering of identi ed interactions was based on the differential gene expression pro le (between AD and control) of sender and receiver cell types.Interactions were retained only if 1) the ligand and receptor genes were expressed in more than 10% of sender and receiver cells, and 2) the gene expression of ligands met an absolute log2 fold change > 0.1 (logFC) with an adjusted p-value < 0.05.

Intracellular target genes analysis of dysregulated intercellular communication signals
We further identi ed the potential targeted genes affected by the dysregulated intercellular signals.
Speci cally, we applied the NicheNet algorithm [25] to infer the regulatory potential of ligands of interest in the sender cells on their potential target genes in the receiver cells.In our analysis, the ligands of the dysregulated LR interactions identi ed in the discovery dataset were used as the ligand of interest for each sender cell population.Differentially expressed genes (DEGs) in AD vs. control in each receiver cell population were used as the potential target gene of interest in the NicheNet analysis.
We identi ed the DEGs in each receiver cell population using the FindMarkers function in the Seurat package with default parameters in the discovery and replication datasets separately.The MAST model was used to obtain the p-values and the adjusted p-values, based on the Bonferroni correction using all genes in the datasets [26].Additionally, sex, age of death, and number of features were included as covariates in the differential analysis.
Following the o cial NicheNet pipeline, we used the predict_ligand_activities function to calculate the ligand activity, which is based on the correlation between prior target gene expression predictions and the observed changes in gene expression.The area under the precision-recall curve (AUPR) was calculated and used to prioritize the ligands.Regulatory potential scores were then calculated between prioritized ligands and potential targeted genes within receiver cells using the default parameters.Active ligandtarget connections were visualized if their regulatory potential score exceeded the 25% quantile of scores of interactions between prioritized ligands and their top targets.Genes expressed in over 10% of receiver cells were considered the background gene.

Gene Ontology (GO) terms ltration
To examine the biological relevance of dysregulated intercellular signals in AD, we conducted pathway enrichment analysis of the genes in the dysregulated LR interactions identi ed in the previous steps.We used 10,532 GO terms from three domains, including Biological Processes (BP), Molecular Functions (MF), and Cellular Components (CC), from the Molecular Signatures Database (MSigDB) (version 2023.1.Hs, accessed on March 6th, 2023) [27].We limited the GO terms to 25 to 500 genes to lter small or large gene set.Only the GO terms containing at least one dysregulated LR gene pair, as identi ed in the preceding analytical step, were retained as the candidate terms for further investigation.

Pathway enrichment analysis using GWAS summary statistics
We implemented the GWAS statistic ne-mapping tool, MAGMA, to detect AD-associated pathways encompassing the dysregulated LR interactions [28].In the past decade, more than six AD GWASs were published [17-20, 29, 30].These studies included shared and distinct participants, allowing for the characterization of new genetic risk factors associated with AD.In our analysis, we used the summary statistic data from a meta-analysis GWAS performed by Wightman et al. [19], including 398,058 individuals (39,918 clinically diagnosed AD cases and 358,140 controls) of European descent, with proxy cases from sources from the UK BioBank (UKBB) and 23andMe excluded [19].
Following the standard pipeline, we rst employed the MAGMA tool to evaluate gene-level signi cance using the collected AD GWAS summary statistics [19].A gene annotation with a 35kb window upstream and a 10kb window downstream was used for the MAGMA gene analyses.Subsequently, we conducted the pathway analysis employing MAGMA to identify AD-associated GO terms containing dysregulated LR interactions [28].The nal results were ltered based on a Benjamini-Hochberg (BH) adjusted p-value < 0.05 [31].

Pathway-based polygenic scores analysis using WGS data
We utilized PRSet, a recently released pathway-based polygenic scores (PGSs) analysis tool, to further evaluate the potential association of GO terms with AD, focusing on those terms encompassing dysregulated LR interactions [32].Brie y, the PRSet method employs a classical clumping + thresholding (C + T) technique to calculate pathway speci c PGSs in relation to selected GO terms for individuals with genotyping data.Single nucleotide polymorphisms (SNPs) falling within regions of interest are preferentially retained for each linkage disequilibrium (LD) clump of SNPs, with a clumping distance of 500kb to either side of the index SNP and an LD r 2 threshold > 0.2.
The identical AD GWAS summary statistics from the MAGMA analysis and the gene coordinates of each gene were used as the base data in the PRSet analysis [19].In addition, a WGS dataset of 1,894 individuals of AD patients and controls, downloaded from the AD Knowledge Portal, was used for pathway speci c PGSs evaluation [23,33].The WGS dataset includes participants from three large cohorts: 1,200 individuals from ROSMAP [34], 354 from the Mount Sinai Brain Bank (MSBB) [35], and 350 from Mayo Clinic [36].The original WGS data were aligned to the human reference GRCh37 and processed using the GATK best practices work ow [33].The dataset was subsequently re ned based on race, resulting in 1,746 individuals of European descent for the analysis.
The performance of the generated PGS for each GO term was initially determined through a generalized linear model.The covariates used in the model were sex, age at death, the number of APOE4 alleles, and the rst ten principal components (PC).We used PRSet competitive p-values calculation, based on permutation test, to test for signal enrichment compared to identically clumped SNPs in regions of the genome considered background (all genes).A pathway PGS with competitive p-values ≤ 0.05 was considered signi cantly enriched in AD.

Drug repurposing analysis
Based on the drug target analysis strategies from our previous work [37], we identi ed drugs that could potentially be repurposed to target genes involved in dysregulated LR interactions in AD.Here, we employed the Therapeutic Target Database (TTD) to obtain information on drugs and their corresponding investigational, literature-curated, and FDA-approved targets [38].We then prioritized candidate repurposing drugs and compounds based on their ability to cross the blood-brain barrier (BBB) based on existing literature [38,39].

Identi cation of dysregulated intercellular networks in AD
The integrative analysis framework is depicted in Fig. 1.To systematically examine the intercellular communication signals in healthy controls and AD, we analyzed two snRNA-seq datasets of postmortem PFC samples [8, 10], utilizing one for discovery and the other for replication, as stated in the Materials and methods section.The discovery dataset was derived from postmortem PFC samples of 24 AD participants and 24 age of death and sex matched controls from the ROSMAP cohort [8].The replication dataset was derived from postmortem PFC tissue from 11 AD participants and seven age of deathmatched controls from UCI MIND-ADRC [10].After performing universal preprocessing and quality control procedures, we analyzed 69,499 nuclei from the discovery dataset and 56,440 nuclei from the replication dataset, corresponding to six major brain cell types, namely astrocytes, excitatory and inhibitory neurons, microglia, oligodendrocytes and OPCs (Additional le 1: Fig. S1).We identi ed intercellular communication signals using CellChat v.1.6.0 [11], based on an updated resource comprising 4,701 consensus LR pairs [12].We then predicted intercellular communication signals separately for AD and controls in the discovery and replication datasets.
In the discovery dataset, we identi ed 987 and 1,211 LR interactions (permutation p-value < 0.05) across cell type pairs in AD and controls (Fig. 2a).Moreover, we found a decrease in interaction strength within the AD group (64.804) than in controls (76.541), which was computed by summing the communication probabilities of all inferred LR pairs (Fig. 2a).This suggests a general decline in intercellular communication in AD.Focusing on cell-type-speci c communication alterations, we found that both outgoing and incoming intercellular communication signals in excitatory and inhibitory neurons exhibited decreases in both quantity and strength in AD samples (Additional le 1: Fig. S2a).In non-neuronal cell types, astrocytes and OPCs showed decreased incoming and outgoing communications connecting with neuronal cell types.On the other hand, we found that microglia showed a mixed pattern in AD, with a higher level of outgoing communication with astrocytes, but a lower level of outgoing with neuronal cell types (Additional le 1: Fig. S2a).
Non-neuronal cell type mediated dysregulation in LR interactions revealed neuroin ammation and calcium dyshomeostasis in AD Our investigation delved into the alterations in each LR gene pair, aiming to nd the dysregulated LR interactions that may be driving the intercellular communication disruption in AD.In total, we identi ed 316 dysregulated LR interactions across six major brain cell types (Fig. 2b, c, Additional le 2: Table S1), de ned as the LR interactions (permutation p-value < 0.05) that exhibited different communication probabilities and had genes encoding ligands differentially expressed in AD (Materials and Methods).
Among them, 58 LR interactions were upregulated, and 258 of them were downregulated in AD (Fig. 2b).Our analysis in the discovery dataset revealed complex intercellular communication patterns across various sender and receiver cell types.Notable interactions occurred in the astrocytes-to-neurons, between excitatory and inhibitory neurons, and in the microglia-to-astrocytes signaling pathways (Fig. 2b, c).
In astrocytes-to-neurons signaling, we found majorly decreased intercellular signals from astrocytes to the two major neuronal cell types.The dysregulated LR interactions involve known AD risk genes as ligands or receptors, such as APOE-LRP1 and APOE-SORL1.The APOE-LRP1 interaction is known to mediate the clearance of β-amyloid across the BBB, thereby regulating β-amyloid transcytosis from the brain to the periphery.Targeting this pair has been suggested as a potential AD treatment [40,41].The receptor encoded by SORL1 has been implicated in β-amyloid clearance [42].In addition to pinpointing well-known AD-associated intercellular signals, our analysis identi ed potential novel downregulated LR interactions in AD, such as PTN-PTPRS and PTN-CHD10.Pleiotrophin (PTN) is a heparin-binding growth factor that regulates peripheral and central immune responses.The interaction of PTN and PTPRS has been reported to play a role in neuroin ammation, an important component in AD [43].Furthermore, we detected downregulation in the communication signaling between excitatory and inhibitory neurons (Fig. 2c, Additional le 1: Fig. S2c).The implicated genes encoding ligands calmodulin (CALM), speci cally CALM1 and CALM3, were present in 25 downregulated LR interactions between excitatory neurons and inhibitory neurons.Our analysis also revealed downregulated CALM signals from neurons to non-neuronal cell types, such as astrocytes and OPCs.This nding indicates a potential association between calcium ion channel dysfunction, calcium dyshomeostasis, and AD pathology [44].In addition, NRXN1, encoding a presynaptic cell adhesion molecule that interacts with Neuroligin 1 (NLGN1), was downregulated in our analyses.Notably, NLGN1, which modulates the toxicity of β-amyloid oligomers, was observed to be altered in the hippocampus of AD individuals [45][46][47][48].

Replication analysis of dysregulated intercellular communication in AD
To increase the validity of the results, we replicated the analysis using an independent snRNA-seq dataset (n = 56,440 nuclei) from UCI MIND-ADRC [10].We successfully replicated 210 out of 316 dysregulated LR interactions across the six major cell types (Fig. 2d, Additional le 3: Table S2).We found consistently downregulated LR interactions in astrocytes-to-neurons and between excitatory and inhibitory neurons signaling.Despite identifying the same dysregulated LR interaction in microglia-to-astrocytes signaling, we failed to replicate the direction of these interactions.However, the LR interaction, HSP90AA1-EGFR showed consistent upregulation from the microglia to OPC.The EGFR signaling pathway is known to be associated with AD, and recent studies suggest that EGFR inhibitors can have potential bene cial effects in mitigating pathological sequelae in AD [51].

Dysregulated intercellular signaling pathways
Our comparative analysis identi ed dynamic communication patterns at the signaling pathway level (Additional le 1: Fig. S2e-f), revealing distinct changes in information ow between AD and control subjects.Most identi ed pathways exhibited decreased signaling strength in AD across various cell types.Notably, the outgoing and incoming signaling strengths of the IGF pathway were downregulated in AD in astrocytes, excitatory and inhibitory neurons (Additional le 1: Fig. S2e-f).Interestingly, the two wellknown AD-associated pathways, APOE and PSAP signaling pathways, demonstrated a similar ow of information between conditions, albeit with a reduced signaling strength among astrocytes (Additional le 1: Fig. S2d).This suggests a need for further examination of individual LR pairs within each signaling pathway.

Intracellular communication analysis revealed the regulatory potential of dysregulated ligand genes on the target genes in AD
To identify the potential path from dysregulated intercellular signals to potential target genes, we further examined the regulatory potential of the ligand genes in dysregulated LR interactions in DEGs in receiver cells.We evaluated potential connections on expression pro les based on prior knowledge of ligandtarget links using the NicheNet R package [25].
Ligand genes of downregulated LR pairs from astrocytes to excitatory neurons, such as AGT, NRXN1, APOE, RTN4, and PTN, exhibited increased ligand activity but relatively low expression in AD (Fig. 3a, b).Notably, angiotensinogen encoded by AGT displayed the most substantial potential regulatory in uence on DEGs in excitatory neurons, including FILIPL1, SYNPO, and MAPK3 (Fig. 3b).Additionally, ligand-target gene pairs, such as APOE and PTN linked to CIRBP and FTH1, respectively, were replicated in the replication dataset.We performed an over-representation analysis on genes involved in dysregulated LR interactions and predicted target genes to further elucidate underlying biological functions.The results indicated signi cant enrichment in the regulation of the nervous system and neuron projection developments (adjusted p-values < 0.05, Fig. 3c, g).
We next explored the regulatory potential of dysregulated ligand genes in inhibitory neurons on the DEGs of excitatory neurons.Ligands encoded by genes, such as NRXN1, RTN4, CALM1, and CALM3, exhibited high ligand activity, potentially modulating the predicted target genes in excitatory neurons (Fig. 3d, e).
Over-representation analysis of dysregulated LR gene pairs and predicted target genes between inhibitory and excitatory neurons yielded signi cant ndings, including regulation of ion transmembrane transport, membrane potentials, and cyclase activity (Fig. 3f).These ndings suggest a critical role of transmembrane signaling dysregulation in AD pathology.

Integrative pathway-level analyses revealed AD-associated GO terms encompassing dysregulated LR interactions
Our intercellular communication analyses yielded hundreds of dysregulated LR interactions across six major brain cell types (Fig. 1b).To gain a more profound understanding of the biological relevance of dysregulated intercellular signaling in AD, we performed pathway analyses by incorporating AD GWAS summary statistics and WGS data.We downloaded all GO terms from three domains (BP, MF, and CC) from MSigDB (version 2023.1.Hs, accessed on March 6th, 2023) [27].We found 298 GO terms that contain at least one dysregulated LR gene pair that were identi ed and replicated in intercellular communication analyses in both discovery and replication snRNA-seq datasets.
We rst applied MAGMA to leverage AD GWAS summary statistics [19] to identify AD-associated GO terms identi ed above.We found a total of 11 GO (6 BP and 5 MF) terms signi cantly associated with AD (adjusted p-values < 0.05, Fig. 4a, Additional le 4: Table S3).The most signi cant GO BP was related to 'amyloid precursor protein metabolic and catabolic processes' (adjusted p-values < 0.01).Furthermore, the MAGMA pathway analysis highlighted several GO MF terms, including 'amide binding', 'peptide binding', and 'amyloid-beta binding' (adjusted p-values < 0.01).The LR gene pair APOE-SORL1 was consistently present across most of the signi cant GO terms identi ed by MAGMA.Interestingly, regulation of calcium ion transport, harboring CALM1 and CALM3-related dysregulated LR pairs, was identi ed as signi cantly associated with AD.In total, six LR interactions were validated by MAGMA (Additional le 4: Table S3).
Pathway-level PGSs have been suggested to better inform disease biology compared to classical PGSs [32]; therefore, we performed an pathway-level PGS analysis utilizing the PRSet tool on candidate GO terms containing dysregulated LR interactions [32].We used the same AD GWAS summary statistics [19] as the based for PGS calculation, and then the WGS data of 1,746 individuals of European descent from three AD cohorts [34][35][36] to evaluate PGS performance (Materials and Methods).Our analysis revealed the PGSs of 58 GO terms signi cantly associated with AD (competitive p-values < 0.05, Additional le 5: Table S4).Figure 3b shows the top 15 GO terms associated with AD, primarily centered on endocytosisrelated cellular components, such as endocytic vesicle and endocytic vesicle membrane.Additionally, the PRSet analysis indicated a signi cant enrichment of 'amyloid precursor protein catabolic and metabolic processes' in AD.
We identi ed 21 LR interactions signi cantly associated with AD supported by either MAGMA or pathwaylevel PGS analysis (Additional le 6: Table S5).Of these, six LR interactions were highlighted by both analyses, including APOE-SORL1, APOE-LRP1, CALM1-CACNA1C, CALM1-RYR2, CALM3-CACNA1C, CALM3-RYR2 (Fig. 4c). Figure 4d illustrates that the LR pair APOE-SORL1 was downregulated in astrocytes-to-neurons and in microglia-to-neurons signaling.Moreover, the intercellular signaling from neuronal cells to other non-neuronal cell types, mediated by the ligands encoded by CALM1 and CALM3 and their receptors encoded by CACNA1C and RYR2, displayed a downregulated trend.

Prioritization of repurposable drug targeting dysregulated cell-cell communication signals in AD disease
Finally, following our previous work, we explored potential repurposable drugs targeting the 21 highcon dent dysregulated LR pairs [37].By inquiring the TTD, we identi ed 14 FDA-approved drugs capable of crossing the BBB [38,39].These drugs target one ligand (HSP90AA1) and four receptors (EGFR, CACNA1C, ALK, INSR) within the dysregulated LR interactions (Table 1).Additionally, four receptors in these pairs were found to be targets of either investigational drugs or previously reported drugs in the literature (Table 1), including GRM5, GRM7, LRP1, and APOE.GRM5 refers to the metabotropic glutamate receptor 5 (mGluR5), targeted by the drug ADX-48621, which is currently being investigated for Parkinson's disease, dyskinesia, and mood disorders (https://clinicaltrials.gov/, NCT04857359).GRM7 refers to the metabotropic glutamate receptor 7 (mGluR7) and is targeted by the drug MPPG, which is still a discovery agent [52].Currently, there is no drug targeting LRP1 under investigation.For APOE, the drug AEM-28 is under study for hyperlipidemia [38].

Discussion
In this study, we integrated human brain snRNA-seq datasets, GWAS summary statistics and WGS from AD and control individuals to identify cell type-speci c dysregulated LR pairs and their underlying biological pathways.We identi ed key known and potential novel dysregulated LR interactions and highlighted vulnerable cell types in AD.Our pathway analyses further prioritized dysregulated LR interactions and related biological pathways supported by genetic association data.Our analysis provides a detailed landscape of cellular communication alterations in AD (Fig. 5), highlighting the power of multi-layered data integration in the study of complex diseases.
Our integrative analysis revealed the critical role of dysregulated astrocytes-to-neurons signaling and related biological functions associated with AD.Our comprehensive bioinformatics analysis highlights that the well-known gene APOE, which encodes the ligand in three dysregulated LR pairs, interacts with receptors encoded by LRP1, LRP4, and SORL1 (Fig. 2a).In addition, LR pairs involving APOE were found to be implicated in top enriched GO terms in our analyses, such as 'endocytic vesicle' and 'negative regulation of amyloid precursor protein catabolic process' (Fig. 4b).These ndings underscore the central role of APOE signaling in the interplay between non-neurons and neurons in the pathophysiology of AD [27,28].In addition, pleiotrophin, encoded by PTN, is a heparin-binding growth factor that regulates peripheral and central immune responses.We found that PTN-involved LR interactions (PTN-PTPRZ1 and PTN-PTPRB) were downregulated from astrocytes to excitatory and inhibitory neurons.The interactions of PTN with protein tyrosine phosphatase receptor type Z polypeptide 1 (PTPRZ1) and protein tyrosine phosphatase receptor type S polypeptide (PTPRB) may play a role in cell proliferation and regulation, both of which are important in AD [43].
Our analysis underscores the pivotal role of calcium dyshomeostasis in the pathogenesis of AD.Notably, CALM, encoded by CALM1 and CALM3, served as a ligand in 25 downregulated LR pairs between excitatory and inhibitory neurons in AD.These LR pairs displayed alterations between excitatory and inhibitory neurons in our analysis.Among them, ten LR gene pairs (CALM1-GRM5, CALM1-GRM7, CALM1-RYR2, CALM -GRM5, and CALM3-GRM7, CALM3-RYR2, CALM1-CACNA1C, CALM3-CACNA1C, CALM3-EGFR, CALM3-INSR) were prioritized in the pathway analyses.Interestingly, the metabotropic glutamate receptor (GRM) was found to be the receptor in ve of these 25 pairs.In general, CALMs interact with GRMs to regulate synaptic plasticity.GRM5 gene is ubiquitously expressed in brain regions implicated in AD phenotypes in mice and in regions linked to memory and learning [53,54].Our pathway analyses highlighted biological functions-such as regulation of calcium ion transport, second messengermediated signaling, and maintenance of location, which encompass four dysregulated LR pairs, including CALM1-RYR2, CALM3-RYR2, CALM1-CACNA1C, CALM3-CACNA1C (Fig. 4, Additional le 6: Table S5).RYR2 is a receptor to CALM1, and the binding of CALM1 to RYR2 has been shown to limit neuronal loss in AD [55].Voltage-dependent L-type calcium channel subunit alpha-1C (CACNA1C) interacts with CALM1 and CALM3 to regulate calcium in ux, and it can be related to neuronal survival and synaptic e ciency, and is thought to be involved in attention, learning, memory, and stress response [56-59].
Our ligand-target gene analysis revealed the potential regulatory role of ligands encoded by CALM1 and CALM3 on the DEGs in excitatory neurons.The predicted target genes, CIRBP and FTH1, were replicated in the independent dataset.Cold-inducible RNA-binding protein (CIRBP) is a general stress-response protein, which was downregulated in AD in our analysis (Fig. 3b).It has been proposed that CRIBP exerts a protective effect against neuronal amyloid toxicity via antioxidative and antiapoptotic pathways [60].
The dysregulation of ferritin heavy chain 1 (FTH1), on the other hand, is linked with neuronal death and memory impairments through iron dyshomeostasis [61].
In our analysis, most intercellular signals mediated by LR pairs were downregulated across six major cell types in AD.Notably, we observed upregulated LR interactions from microglia to astrocytes in the discovery dataset, although this was not replicated in the independent replication dataset.C3 was found altered as a ligand in two different LR pairs, C3-LRP1 and C3-CD81.Both pairs were upregulated in microglia, astrocytes, and OPCs, with microglia as the sender and astrocytes and OPCs as the receivers (Fig. 2c).C3 is a protein that is part of the complement system and part of the immune system; it colocalizes with amyloid plaques in AD.Low-density lipoprotein receptor-related protein 1 (LRP1) is a surface receptor and mediates pathways that interact with astrocytes and pericytes, the last of which is associated with the BBB.LRP1 expression is known to decrease in endothelial cells due to normal aging and in AD.C3 interacts and can bind with low-density LRP1 to regulate immune response and participate in several cellular processes [41,[62][63][64][65]. Ligand C3 and receptor CD81 play an inhibitory role in the control of immune responses [49].We also identi ed alpha-2-macroglobulin (A2M) as a ligand in the A2M-LRP1 pair, which was upregulated in microglia.A2M interacts with LRP1 to regulate cholesterol metabolism and is considered a potential therapeutic target in AD [62].Our ligand-target gene analysis from microglia to astrocytes suggests the regulatory potential of ligands encoded by A2M and C3 on the DEGs in the receiver cells (Additional le 1: Fig. S3a, b).Over-representation analysis on genes involved in dysregulated LR pairs and predicted target genes indicated signi cant enrichment in 'amyloid-beta clearance' and functions related to regulation of lipid (Additional le 1: Fig. S3c).
Moreover, two identi ed pairs, NRXN1-NLGN1 and NRXN1-NLGN3, are related to neurexins (NRXNs) and neuroligins (NLGNs) and their signaling is decreased in AD in a myriad of cell types, including astrocytes, excitatory and inhibitory neurons.NRXNs are cell-surface receptors that bind NLGNs, forming a crucial transsynaptic complex at brain synapses.This transsynaptic complex is vital for e cient neurotransmission and is involved in forming synaptic contacts and functional synaptic structures.
Recent reports suggest that NRXNs and NLGNs undergo proteolytic processing by presenilins at synapses, a mechanism implicated in AD, suggesting a potential dysfunction in the NRXN-NLGN pathway in AD pathology [45].
Further, we observed upregulation of other LR pairs, including PSAP-LRP1 and PSAP-GPR37, in astrocytes, microglia, and oligodendrocytes (Fig. 2c, Additional le 2: Table S1).Prosaposin (PSAP) is a highly conserved glycoprotein that is a precursor of saposins; it also serves as a neurotrophic factor and a regulator of lysosomal enzymes.PSAP is known to interact with LRP1 in AD, with the interaction between PSAP and LRP1 being involved in the regulation of amyloid-beta metabolism [66].The expression of PSAP and its receptor GPR37 is upregulated in the hippocampus of individuals with AD [67-69].
Finally, other LR pairs possibly related to AD involved genes that encode receptors, such as epidermal growth factor receptor (EGFR), insulin receptor (INSR), corticotropin-releasing hormone receptor 1 (CRHR1), and adenylate cyclase-activating polypeptide type I receptor (ADCYAP1R1) (Additional le 1: Fig. S2b, c).In general, they are involved in cell proliferation and differentiation, glucose metabolism, and stress response [70].In addition, EGFR has been identi ed as the receptor in two upregulated LR pairs, involving heat shock protein 90 alpha family class A member 1 (HSP90AA1) and neuregulin 3 (NRG3) as the ligands.Both are implicated in cell proliferation and differentiation; NRG3 has been implicated in cognitive impairment [71,72].INSR was also found as a gene that encodes the receptor for sorbin and SH3 domain-containing protein 1 (SORBS1), downregulated in astrocytes, excitatory neurons, inhibitory neurons, and oligodendrocytes; the SORBS1-INSR is known to regulate glucose metabolism.Moreover, we found that the GO BP term 'regulation of cellular and carbohydrate metabolic process' encompassing SORBS1-INSR was associated with AD (Additional le 6: Table S5).
Our drug target analysis revealed existing and potentially novel therapeutic targets of dysregulated LR pairs in AD.Regarding EGFR, erlotinib, ge tinib, and osimertinib were found as potential drugs for repurposing.Both erlotinib and osimertinib are used to treat lung and pancreatic cancers and can cross the BBB (Table 1).They are tyrosine kinase inhibitors that work by blocking the kinase activity of EGFR, which is involved in cell growth and survival [73].Erlotinib and ge tinib also have antioxidant properties [74].It has been hypothesized that both drugs may enhance axon regeneration after neurodegeneration [51].In addition, receptor ALK (in the PTN-ALK LR interaction) was targeted by four potential repurposable drugs that cross the BBB, including alectinib, ceritinib, entrectinib, and lorlatinib (Table 1 While our integrative study used multiple, large-scale datasets, there were several limitations.First, our inference of dysregulated LR interaction was primarily dependent on the completeness of snRNA-seq datasets, cell type annotation, and the reliability of the LR dataset.Despite employing one of the most comprehensive snRNA-seq datasets of AD and controls currently available [8], we limited our analysis to six major cell types due to a relatively low cell count of pericytes and endothelial cells.We also performed replication analysis to ensure the reliability of the analysis.However, more complex intercellular signals could be unveiled in rare cell types or subclasses of major cell types with the employment of larger snRNA-seq datasets.Second, inadequate annotation of intercellular signaling pathways and intracellular regulatory networks may impede our pathway analyses of dysregulated LR pairs in AD.To address this point, we utilized comprehensive GO gene sets to evaluate the biological functions in uenced by dysregulated LR signals in AD.Third, our cell-cell communication analysis was limited to the PFC region.
Considering that AD pathology affects multiple brain regions, including the entorhinal cortex and hippocampus, further investigations across multiple brain regions are necessary for a more in-depth understanding of region-speci c dysregulated intercellular signals in AD.Finally, rigorous laboratory experimental validation, which we did not perform because it was outside the scope of this study, will further validate the causal relationships between identi ed dysregulated intercellular interactions and disease progression.

Conclusions
Our comprehensive in silico investigation provides novel insights into the complex intercellular signaling dynamics underpinning AD.By applying a novel analysis pipeline integrating snRNA-seq, GWAS, and   Figure 4

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Table 1
Drug repurposing analysis of Therapeutic Target Database This compound has acetylcholinesterase (AChE) inhibitory activities, a mechanism that has been proposed to treat AD [82] and has shown neuroprotective activity.Recently, brain insulin resistance has been found to play a role in normal memory processes and insulin irregularities may contribute to cognitive and brain changes associated with AD[83].Metformin and insulin target the INSR and appeared as potentially repurposable drugs in our analyses.Evidence from clinical studies has demonstrated that metformin use contributes to a lower risk of developing AD and better cognitive performance[84].Intranasally administered insulin is assumed to trigger improvements in synaptic plasticity, regional glucose uptake, and alleviations of AD neuropathology.Pilot clinical trials of intranasal insulin administration in individuals with mild cognitive impairment or AD indicate that acute and prolonged intranasal insulin administration can enhance memory performance[85].
[79,o called cromolyn).Both have anti-in ammatory properties, with cromolyn speci cally reducing neuroin ammation.Cromolyn has been proposed as a new therapeutic target for AD[78].Cromolyn has been shown to reduce levels of amyloid beta by promoting microglial phagocytosis[79, 80].It also reduces the secretion of in ammatory cytokines by the microglia [81], reducing neuroin ammation in neural cells.The root of Rauwol a serpentina, currently a discovery agent, targets the receptor CACNA1C.